Antithrombin nucleotides and proteins from horn fly

ABSTRACT

Compositions and methods for preventing hematophagous infestation of cattle are provided, directed at isolated proteins with antithrombin activity and nucleotide sequences encoding the proteins. The protein named thrombostasin is isolated from the salivary glands of  Haematobia irritans . The compositions are useful as veterinary vaccines in prevention of blood-feeding in cattle by the infesting horn fly. The proteins of the invention are also useful in treatment of thrombosis.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No. 09/376,113filed Aug. 17, 1999, now issued as U.S. Pat. No. 6,451,992, which claimsthe benefit of U.S. Provisional Application No. 60/097,227, filed Aug.20, 1998, which is hereby incorporated herein in its entire byreference.

FIELD OF THE INVENTION

The invention relates to veterinary vaccines for prevention ofhematophagous infestation of cattle and medical treatment of thrombosis.

BACKGROUND OF THE INVENTION

Losses in livestock production in the United States due to ectoparasiteinfestations have been estimated to exceed $2.26 billion annually(Byford et al. (1992) J. Anim. Sci. 70:597-602). Of the five to sixmajor arthropod pest species involved, the horn fly Haematobia irritanslinnaeus is the most significant and widespread. Its annual economicimpact on cattle production in the U.S.A. has been estimated at $730.3million. In Canada, control of this ectoparasite in cattle productionhas been estimated to reduce losses by $71-107 million per year using1977 dollar values (Haufe and Weintraub (1985) Can. Entomol.117:901-907). Thus in North America, the annual economic impact oncattle production by this blood-sucking fly approaches $1 billion.

Physiological manifestations of hornfly infestation include an increasein heart rates, respiration rates, and rectal temperatures.Additionally, water consumption and urine production are significantlyincreased as well as urinary nitrogen secretion. Blood cortisolconcentrations are also significantly increased. Decreased weight gain,increased activity, and decreased grazing have also been reported.(Schwinghammer et al. (1986) J. Econ. Entomol. 79:1010-1014).

The adult stage of both sexes of H. irritans are obligate ectoparasitesthat blood-feed intermittently during the 24 hours of the day. Unlikeother dipterous pests that are transient blood-feeders, (black flies,mosquitoes, horse flies, stable flies), the winged adults of H. irritansremain on the bovine host and, when needing nourishment, recurrentlyinsert their mouthparts into the skin to feed. Harris et al. (1974) Ann.Entomol. Soc. Am. 67:891-894, noted that under experimental conditions,female horn flies spent an average of 163 minutes/day feeding; malesaveraged 96 minutes per day. Each female ingested an average of 17.1 mgof blood per day while males imbibed 12.1 mg/individual due to thedifference in feeding times (Harris and Frazer (1970) Ann. Entomol. Soc.Am. 63:1475-1476).

The scientific literature describing the salivary gland physiology of H.irritans, particularly with reference to blood-feeding, is sparse. Horiet al. (1981) Appl. Ent. Zool, 16:16-23, has compared several categoriesof digestive enzymes in the gut and salivary glands of H. irritans withStomoxys calcitrans (Linnaeus), the stable fly. Weak aminopeptidaseactivity was detected in H. irritans saliva, suggesting that proteasesand glycosidases in the gut are exclusively responsible for digestion ofblood.

The horn fly Haematobia irritans linnaeus is a subspecies with H. i.exigua de Meijere, the buffalo fly that occurs in Australia andelsewhere in the southern hemisphere. Kerlin and Hughes (1992) Med. Vet.Entomol. 6:121-126, have compared enzymes in the saliva of fourparasitic arthropods—H. irritans exigua, Boophilus microplus(Canestrini), Aedes aegypti (Linnaeus), and Lucilia cuprina (Wiedemann)and noted differences in enzyme profiles of saliva between the fourspecies that apparently reflect their dissimilar feeding strategies.These differences were mainly in the type and levels of glycosidase andprotease activities. H. irritans exigua saliva, collected by serotoninstimulation and then evaluated by SDS polyacrylamide gelelectrophoresis, produced 7-8 bands by silver staining. Apyrase activityin saliva and salivary gland extracts (SGEs) of this species wasmarginally detectable, suggesting that this subspecies does not preventbovine platelet aggregation in the same way as many other blood-feedingarthropods (Ribeiro (1987) Ann. Rev. Entomol. 32:463-478).

Furthermore, investigation of immune response of cattle exposed to H.irritans exigua showed production of high levels of circulatingantibodies to some but not all of the buffalo fly antigens;nevertheless, flies feeding on previously exposed cattle did not exhibithigher mortality than those fed on unexposed cattle. (Kerlin andAllingham (1992) Vet. Parasitol. 43:115-129).

Elucidation of biochemical strategies adopted by blood-feedingarthropods has advanced in the past decade. Although the presence ofanticoagulants in saliva of hematophagous arthropods has been known forat least eight decades, only recently have some of the active componentsbeen purified and their molecular structures defined. It has becomeapparent that coagulation factors such as factors Xa and thrombin(factor II), which occur at a nexus in the coagulation cascade, arefrequently targeted.

Studies of saliva from several species of black flies have suggestedthat specific enzyme targets may be associated with host selection(Abebe et al. (1994)). For example, data for zoophagic species thatprefer cattle indicate that thrombin is an important target moleculewhose inactivation may also prevent irreversible platelet aggregation inaddition to impeding the coagulation cascade. See Hudson (1964) Can. J.Zool. 42:113-120, for Stomoxys calcitrans; and Parker and Mant (1979)Thrombos. Haemostas (Stuttg.) 42:743-751, on G. morsitans (Westwood)saliva.

Because of the adverse impact of the above-described ectoparasiticinfestation in cattle, there is a therapeutic and economic need forpreventing such infestation.

There is also need for treatment of thromboembolic diseases.Thromboembolic diseases are among the most important circulatorydiseases. A thrombus is a blood clot that partially or completely blocksblood flow through a blood vessel. An embolus is a thrombus that hasformed elsewhere in the body, broken free, and traveled to the sitewhere blockage occurs. Blockage in the brain results in a stroke, i.e.,a cerebral infarction, a localized area of dead cells. An embolus in alung can produce pulmonary embolism, one of the principal lung diseasesin bed-ridden patients. Bed ridden and elderly persons are alsoparticularly prone to thrombophlebitis, which is a blockage ofcirculation in a leg caused by an embolus. An embolus or thrombuslodging in one of the blood vessels serving the heart causes necrosis ofpart of the heart tissue, a myocardial infarction, commonly called aheart attack.

The initiating event of many myocardial infarctions is the hemorrhageinto atherosclerotic plaques. Such hemorrhage often results in theformation of a thrombus (or blood clot) in the coronary artery whichsupplies the infarct zone. This thrombus is composed of a combination offibrin and blood platelets. The formation of a fibrin-platelet clot hasserious clinical ramifications. The degree and duration of the occlusioncaused by the fibrin-platelet clot determines the mass of the infarctzone and the extent of damage.

The formation of fibrin-platelet clots in other parts of the circulatorysystem may be partially prevented through the use of anticoagulants,such as heparin. Unfortunately, heparin has not been found to beuniversally effective in preventing reocclusion in myocardial infarctionvictims in which the degree of blood vessel occlusion is greater than orequal to 70%, particularly in those patients with severe residualcoronary stenosis. Among the more promising of the agents are hirudinand its analogs, which bind to and inactivate thrombin. Hirudin has atheoretical advantage over heparin as an anti-thrombotic agent. Thrombinbound to thrombi or platelets is relatively protected from inhibition byheparin while hirudin, at least in vitro, is still effective. Otherpromising investigational agents include fibrinogen receptorantagonists, which block platelet aggregation and dense granule releaseby a mechanism distinct from that of aspirin, and inhibitors ofthromboxane production.

There is therefore a need for additional antithrombin agents whichexhibit low toxicity, little or no antigenicity, and a very shortclearance time from circulation.

SUMMARY OF THE INVENTION

Isolated proteins with antithrombin activity and nucleotide sequencesencoding the proteins are provided. The protein named thrombostasin isisolated from the salivary glands of Haematobia irritans, theblood-feeding horn fly. The provided proteins and nucleotides areparticularly useful as veterinary vaccines in prevention ofblood-feeding in cattle by the infesting horn fly.

The proteins of the invention are also useful in treatment ofthrombosis.

Methods of administering the proteins and nucleotide sequences of theinvention are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows molecular weight comparison of proteins in colony-versusfield collected flies by relative mobility on SDS PAGE.

FIG. 2 depicts the recalcification time assay to test for anti coagulantactivity in H. irritans saliva.

FIG. 3 shows the effect of H. irritans saliva on clotting of Factor IIdeficient plasma.

FIG. 4 shows inhibition of thrombin hydrolysis of S238 by H. irritanssaliva.

FIGS. 5A, 5B, and 5C show HPLC purification of active salivarythrombostasin.

FIG. 6 shows SDS PAGE profile of HPLC purified salivary anticlottingprotein thrombostasin.

DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions for preventing hematophagy (blood-feeding) incattle, and treatment of thrombosis in a mammal are provided. Thecompositions comprise protein from the salivary gland of thehematophagous horn fly Haematobia irritans which, as described in Yeateset al. (1999) Annu. Rev. Entemol. 44: 397-428, belong to the suborderCyclorrhapha of the order Diptera. Nucleotide sequences encoding theantithrombin protein are additionally provided. The protein has beendesignated thrombostasin. The major function of the protein is toprevent coagulation by inhibiting the activity of thrombin (factor II).

By “hematophagy” is intended feeding on the blood of a host organism byanother organism. By “hematophagous infestation” is intended ahost-parasite relationship comprising feeding on the blood of the hostby the parasite. By “thrombosis” is intended the formation, developmentor presence of a thrombus. By “antithrombin activity” is intended abiological activity that reduces or eliminates the procoagulant actionof thrombin; and/or inhibits thrombosis.

It is recognized that methods are available in the art to obtain thecomplete coding sequence for the antithrombin protein of the invention.Such methods are disclosed for example in Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (Cold Spring Harbor LaboratoryPress, Plainview, N.Y.).

Substantially purified preparations of thrombostasin are provided. Suchsubstantially purified preparations include proteins substantially freeof any compound normally associated with the protein in its naturalstate. Such proteins can be assessed for purity by SDS-PAGE,chromatography, electrophoresis or other methods. See, M. P. Deutscher(ed.), Guide to Protein Purification, Academic Press, Inc. (1990).

The terms “substantially pure” or “substantially purified” are not meantto exclude artificial or synthetic mixtures of the protein with othercompounds. It is recognized that the antithrombin proteins of thepresent invention include those proteins homologous to, and havingessentially the same biological properties as, the antithrombin proteindescribed herein, and particularly the protein disclosed herein in SEQID NO: 2, SEQ ID NO:5, or SEQ ID NO:7. This definition is intended toencompass natural allelic variations in the genes. It is also recognizedthat “substantially purified” proteins of the present invention asdescribed herein can be of other species of origin, including but notlimited to other species of the suborder Cyclorrhapha.

The invention also provides fragments of the antithrombin protein andnucleotide sequence disclosed in SEQ ID NOs: 1, 2, 4, 5, 6, and 7.Fragments of the protein may range in size from at least 10, 20, 30 ormore amino acids. Such fragments may retain biological activity orcomprise active regions of the protein.

Polynucleotide fragments may also range in size from at least 15, 20, 30or more contiguous nucleotides. The sequences find use as hybridizationprocess or molecular markers.

Such fragments can be readily made by chemical methods includingcommercially available automated methods or by recombinant DNA methodsknown to the ordinarily skilled artisan, and described below. It isrecognized that biological functions of anti-hemostasis, including thoserelated to antithrombin anticoagulant activity and/or modulation ofimmune response may be carried out by the described fragments.

The invention additionally encompasses the nucleotide sequences whichencode the proteins of the invention. The nucleotide sequence of thePCR-cloned coding sequence from H. irritans is provided in SEQ ID NO: 1;however, it is recognized that cloned genes of the present invention canbe of other species of origin, including but not limited to otherspecies of the suborder Cyclorrhapha.

DNAs which hybridize to the nucleotide sequence of the antithrombin genefrom the horn fly are also an aspect of this invention. Conditions,which will permit other DNAs to hybridize to the DNA disclosed herein,can be determined in accordance with known techniques. For example,hybridization of such sequences may be carried out under conditions ofreduced stringency, medium stringency or even stringent conditions(e.g., conditions represented by a wash stringency of 35-40% Formamidewith 5× Denhardt's solution, 0.5% SDS and 1×SSPE at 37° C.; conditionsrepresented by a wash stringency of 40-45% Formamide with 5×Denhardt'ssolution, 0.5% SDS, and 1× SSPE at 42EC; and conditions represented by awash stringency of 50% Formamide with 5× Denhardt's solution, 0.5% SDSand 1×SSPE at 42EC, respectively, to DNA encoding the genes disclosedherein in a standard hybridization assay. See J. Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2^(nd) ed.) (Cold Spring HarborLaboratory).

In general, sequences which code for the antithrombin protein andhybridize to the nucleotide sequence disclosed herein will be at least40% homologous, about 60% to 70% homologous, and even about 80%, 85%,90% homologous or more with the disclosed sequences. Such sequences aresubstantially homologous to the nucleotide sequences disclosed hereinand encompassed by the invention. Further, the amino acid sequences ofthe antithrombin proteins isolated by hybridization to the DNA'sdisclosed herein are also an aspect of this invention. The degeneracy ofthe genetic code, which allows different nucleic acid sequences to codefor the same protein or peptide, is well known in the literature. See,e.g., U.S. Pat. No. 4,757,006.

The hybridization probes may be cDNA fragments or oligonucleotides, andmay be labeled with a detectable group as known in the art. Pairs ofprobes which will serve as PCR primers for the antithrombin gene or aprotein thereof may be used in accordance with the process described inU.S. Pat. Nos. 4,683,202 and 4,683,195.

The polypeptides of the invention may be subject to one or morepost-translational modifications such as sulphation, COOH-amidation,acylation or chemical alteration of the polypeptide chain.

It is recognized that the nucleotide and peptide sequences of theinvention may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions. Methods for suchmanipulations are generally known in the art. For example, amino acidsequence variants of the peptides and proteins can be prepared bymutations in the DNA. Methods for mutagenesis and nucleotide sequencealterations are well known in the art. See, for example, Kunkel, T.(1985) Proc. Natl. Acad. Sci. U.S.A 82:488-492; Kunkel et al. (1987)Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker andGaastra (eds.) Techniques in Molecular Biology, MacMillan PublishingCompany, NY (1983) and the references cited therein. Thus, thenucleotide sequences of the invention include both the naturallyoccurring sequences as well as mutant. Likewise, the peptides andproteins of the invention encompass both naturally occurring andmodified forms thereof. Such variants will continue to possess thedesired activity. It is recognized that the mutations that will be madein the DNA encoding the variant must not place the sequence out ofreading frame and preferably will not create sequences deleterious toexpression of the gene product. See, EP Patent Application, PublicationNo. 75,444.

The proteins of the invention include the naturally occurring forms aswell as variants thereof. These variants will be substantiallyhomologous and functionally equivalent to the native protein. As usedherein, two proteins (or a region of the proteins) are “substantiallyhomologous” when the amino acid sequences are typically at least about40%, more typically at least about 60%-70%, and most typically at leastabout 80%, 85%, 90% or more identical. A substantially homologous aminoacid sequence, according to the present invention, will be encoded by anucleic acid sequence hybridizing to the nucleic acid sequence, orportion thereof, of the nucleotide sequence shown in SEQ ID NO:1, SEQ IDNO:4, SEQ ID NO:6, or otherwise described herein under stringentconditions as more fully described below.

Thus, a variant of a native protein is “substantially homologous” to thenative protein when at least about 40%, more preferably at least about60%-70%, and most preferably at least about 80%, 85%, 90%, or more ofits amino acid sequence is identical to the amino acid sequence of thenative protein. A variant may differ by as few as 1, 2, 3, or 4 aminoacids. A variant polypeptide can differ in amino acid sequence by one ormore substitutions, deletions, insertions, inversions, fusions, andtruncations or a combination of any of these.

By “functionally equivalent” is intended that the sequence of thevariant defines a chain that produces a protein having substantially thesame biological activity as the native protein of interest. Suchfunctionally equivalent variants that comprise substantial sequencevariations are also encompassed by the invention. Thus a functionallyequivalent variant of the native protein will have a sufficientbiological activity to be therapeutically useful. By therapeuticallyuseful is intended effective in achieving a therapeutic goal asdiscussed below.

Methods are available in the art for determining functional equivalence.Biological activity can be measured using assays specifically designedfor measuring activity of the native protein, including assays describedin the present invention. Additionally, antibodies raised against thebiologically active native protein can be tested for their ability tobind to the functionally equivalent variant, where effective binding isindicative of a protein having conformation similar to that of thenative protein.

Variant polypeptides can be fully functional or can lack function in oneor more activities. Thus, in the present case, variations can affect thefunction, for example, of one or more of the modules, domains, orfunctional subregions of the proteins and polypeptides of the invention.

Fully functional variants typically contain only conservative variationor variation in non-critical residues or in non-critical regions.Functional variants can also contain substitution of similar aminoacids, which result in no change or an insignificant change in function.Alternatively, such substitutions may positively or negatively affectfunction to some degree.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region. As indicated, variants can benaturally-occurring or can be made by recombinant means or chemicalsynthesis to provide useful and novel characteristics for thepolypeptide.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science 244:1081-1085 (1989)). Thelatter procedure introduces single alanine mutations at every residue inthe molecule. The resulting mutant molecules are then tested forbiological activity. Sites that are critical can also be determined bystructural analysis such as crystallization, nuclear magnetic resonanceor photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904(1992); de Vos et al. Science 255:306-312 (1992)).

The invention further encompasses variant polynucleotides, and fragmentsthereof, that differ from the nucleotide sequence shown in SEQ ID NO:1,SEQ ID NO:4, or SEQ ID NO:6, or otherwise described herein, due todegeneracy of the genetic code and thus encode the same protein as thatencoded by the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, orSEQ ID NO:6 or otherwise described herein.

The invention also provides nucleic acid molecules encoding the variantpolypeptides described herein. Such polynucleotides may be naturallyoccurring, such as allelic variants (same locus), homologs (differentlocus), and orthologs (different organism), or may be constructed byrecombinant DNA methods or by chemical synthesis. Such non-naturallyoccurring variants may be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. Accordingly, asdiscussed above, the variants can contain nucleotide substitutions,deletions, inversions and insertions.

Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions.

Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. These variants comprise a nucleotidesequence encoding a protein that is at least typically about 40%, moretypically at least about 60%-70%, and most typically at least about 80%,85%, 90% or more homologous to the nucleotide sequence shown in SEQ IDNO:1, SEQ ID NO:4, SEQ ID NO: 6 or otherwise described herein, or afragment of this sequence. Such nucleic acid molecules can readily beidentified as being able to hybridize under stringent conditions, to thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO: 6 orotherwise described herein, or a fragment of the sequence. It isunderstood that stringent hybridization does not indicate substantialhomology where it is due to general homology, such as poly A sequences,or sequences common to all or most proteins in an organism or class ofproteins.

To determine the percent homology of two amino acid sequences, or of twonucleotide sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of one protein ornucleic acid for optimal alignment with the other protein or nucleicacid). The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in one sequence is occupied by the same amino acid residue ornucleotide as the corresponding position in the other sequence, then themolecules are homologous at that position. As used herein, amino acid ornucleic acid “homology” is equivalent to amino acid or nucleic acid“identity”. The percent homology between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent homology equals the number of identical positions/total numberof positions times 100).

The invention also encompasses proteins or polypeptides having a lowerdegree of identity but having sufficient similarity so as to perform oneor more of the same functions performed by the antithrombin proteinsdescribed herein. Similarity is determined by conserved amino acidsubstitution. Such substitutions are those that substitute the givenamino acid in a polypeptide by another amino acid of likecharacteristics. Conservative substitutions are likely to bephenotypically silent. Guidance concerning which amino acid changes arelikely to be phenotypically silent are found in Bowie et al., Science247:1306-1310 (1990).

Both identity and similarity can be readily calculated (ComputationalMolecular Biology, Lesk, A. M., ed., Oxford University Press, New York,1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991). Preferred computer programmethods to determine identify and similarity between two sequencesinclude, but are not limited to, GCG program package (Devereux, J.(1984) Nuc. Acids Res. 12(1):387), BLASTP, BLASTN, and FASTA (Atschul,S. F. (1990) J. Molec. Biol. 215:403); utilizing the default parametersavailable within the programs. By substantial sequence similarity,identity or homology is intended sequences having at least about 60%,70%, 75%, 80%, 85%, 90%, 95% or more similarity.

DNA sequences can also be synthesized chemically or modified bysite-directed mutagenesis to reflect the codon preference of the hostcell and increase the expression efficiency.

The proteins of the invention can be engineered in accordance with thepresent invention by chemical methods or molecular biology techniques.Molecular biology methods are most convenient since proteins can beengineered by manipulating the DNA sequences encoding them. Genomic DNA,cDNA, synthetic DNA, and any combination thereof may be used for thispurpose. Genomic DNA sequences or cDNA sequences encoding proteins canbe isolated based on the amino acid sequence of proteins or certainprotein properties. Many methods of sequence isolation are known in theart of molecular biology. See particularly Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (Cold Spring Harbor LaboratoryPress, Plainview, N.Y.), herein incorporated by reference.

To produce an antithrombin polypeptide by recombinant DNA technology, agene encoding a polypeptide of the invention is prepared. The DNA codingsequence typically does not contain introns. The DNA sequence isisolated and purified, the gene is inserted in an expression vector ableto drive expression and production of the recombinant product. The DNAsequence may be a cDNA sequence, or alternatively a synthetic DNAsequence. A synthetic gene is typically prepared by chemicallysynthesizing oligonucleotides that, in total, correspond to the desiredgene. The synthesized oligonucleotides are then assembled to obtain thegene.

If desired, the gene sequence may be modified by site-directedmutagenesis to introduce one or more coding changes. Typically, a geneis constructed with restriction sites at each end to facilitate itssubsequent manipulation.

The DNA sequence may be constructed to comprise a leader peptide. Theleader peptide is capable of directing secretion of the polypeptide fromcells in which the polypeptide is to be expressed. The sequence encodingthe leader peptide is typically fused to the 5′-end of the DNA sequenceencoding the polypeptide. Leader sequences are known in the art andinclude the OmpA leader peptide, the leader peptide of vesicularstomatitis virus G protein (VSV G protein). The OmpA leader is usefulwhen expression is in a bacterial host, such as E. coli while the VSVGprotein is useful when expression is in insect cells.

The DNA sequence may be constructed to comprise a cleavable site torelease the polypeptide of the invention. A DNA sequence may be usedwhich encodes a carrier polypeptide sequence fused via a cleavablelinkage to the N-terminus of a polypeptide of the invention. Thecleavable linkage may be one cleavable by cyanogen bromide.

For expression of the polypeptides, an expression vector is constructedwhich comprises a DNA sequence encoding the polypeptide which is capableof expressing the polypeptide in a suitable host. Appropriatetranscriptional and translational control elements are provided,including a promoter for the DNA sequence, a transcriptional terminationsite, and translation start and stop codons. The DNA sequence isprovided in the correct frame such as to enable expression of thepolypeptide to occur in a host compatible with the vector.

The expression vector typically comprises an origin of replication and,if desired, a selectable marker gene such as antibiotic resistance. Theexpression vector may be a plasmid, a virus, particularly a baculovirus,and the like.

Once the nucleotide sequences encoding the antithrombin proteins of theinvention have been isolated, they can be manipulated and used toexpress the protein in a variety of hosts including other organisms,including microorganisms.

Once the nucleotide sequence is identified and known, those skilled inthe art can produce large quantities of the protein for therapeutic use.Accordingly, recombinant protein and methods for producing therecombinant protein are encompassed by the present invention. In thismanner, the nucleotide sequence encoding the antithrombin protein can beutilized in vectors for expression in various types of host cells,including both procaryotes and eucaryotes, to produce large quantitiesof the protein, or active analogues, or fragments thereof, and otherconstructs having antithrombin activity.

Generally, methods for the expression of recombinant DNA are known inthe art. See, for example, Sambrook et al. (1989) Molecular Cloning,Cold Spring Harbor Laboratory. Additionally, host cells and expressionvectors, such as the baculovirus expression described in U.S. Pat. Nos.4,745,051 and 4,879,236. In general, a baculovirus expression vectorcomprises a baculovirus genome containing the gene to be expressedinserted into the polyhedron gene at a position ranging from thepolyhedron transcriptional start signal to the ATG start site and underthe transcriptional control of a baculovirus polyhedron promoter.

A broad variety of suitable procaryotic and microbial vectors areavailable. Likewise, the promoters and other regulatory agents used inexpression of foreign proteins are available in the art. Promoterscommonly used in recombinant microbial expression vectors are known inthe art and include the beta-lictamase (penicillinase) and lactosepromoter systems (Chang et al. (1978) Nature 275:615 and Goeddel et al.(1979) Nature 281:544); A tryptophan (TRP) promoter system (Goeddel etal. (1980) Nucleic Acids Res. 8:4057 and the EPO Application PublicationNo. 36,776); and the Tac promoter (DeBoer et al. (1983) Proc. Natl.Acad. Sci. U.S.A, 80:21). While these are commonly used, other microbialpromoters are available. Details concerning nucleotide sequences of manyhave been published, enabling a skilled worker to operably ligate themto DNA encoding the protein in plasmid or viral vectors. See, forexample, Siedenlist et al. (1980) Cell 20:269.

Eucaryotic host cells such as yeast may be transformed with suitableprotein-encoding vectors. See, e.g., U.S. Pat. No. 4,745,057.Saccharomyces cerevisiae is the most commonly used among lowereukaryotic host microorganisms, although a number of other strains arecommonly available. Yeast vectors may contain an origin of replicationfrom the 2 micron yeast plasmid or an autonomously replicating sequence(ARS), a promoter, DNA encoding the desired protein, sequences forpolyadenylation and transcription termination, and a selection gene. Anexemplary plasmid is YRp7, (Stinchcomb et al. (1979) Nature 282:9;Kingsman et al. (1979) Gene 7:141; Tschemper et al. (1980) Gene 10:157).This plasmid contains the trp1 gene, which provides a selection markerfor a mutant strain of yeast lacking the ability to grow in tryptophan,for example ATCC No. 44076 or PEP4-1 (Jones (1977) Genetics 85:12). Thepresence of the trp1 lesion in the yeast host cell genome then providesan effective environment for detecting transformation by growth in theabsence of tryptophan.

Suitable promoter sequences for use in yeast vectors include thepromoters for metallothionein, alcohol dehydrogenase, adenylate cyclase,3-phosphoglycerate kinase (Hitzeman et al. (1980) J. Biol. Chem.255:2073) and other glycolytic enzymes (Hess et al. (1968) J. Adv.Enzyme Reg. 7:149; and Holland et al. (1978) Biochemistry 17:4900) suchas enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. Suitable vectorsand promoters for use in yeast expression are further described in R.Hitzeman et al. EPO Publn. No. 73,657.

The invention provides antibody preparations that selectively bind theproteins of the invention, or any variants or fragments thereof asdescribed. An antibody is considered to selectively bind, even if italso binds to other proteins that are not substantially homologous withthe antithrombin protein. These other proteins share homology with afragment or domain of the antithrombin protein giving rise to antibodiesthat bind to both proteins by virtue of the homologous sequence. In thisaspect, it is recognized that antibody binding to the antithrombinprotein is still selective.

The preparations encompass monoclonal or polyclonal antibodies, intactantibodies or fragments thereof (e.g. Fab), purified preparations suchas affinity-purified preparations, or less pure preparations such asascites fluid, sera and the like. Methods for raising antibodies arewell known in the art and include but are not limited to those describedin Harlow and Lane ((1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory Press), the contents of which are herein incorporatedby reference. The invention also embodies antibody preparations whichneutralize biological functions of the provided proteins, variants orfragments thereof. Such functions include but are not limited toantithrombin activity. The invention also provides compositions capableof modulating the immune response. By modulating the immune response isintended a determinable change in the immune system of a host organismeffected by administering the herein described compositions of theinvention to that host. Working examples of such modulation of immuneresponse, as well as methods of making and assessing selectivity ofantibody preparations are provided in the Experimental section of thisapplication, and are herein incorporated by reference.

The compositions of the present invention find therapeutic use asveterinary vaccines in treatment of hematophagy in a mammal. The methodscomprise administering to the mammal a veterinary vaccine comprising atherapeutically effective amount of the compositions of the invention.In this aspect, a therapeutically effective amount is intended as thatamount which effects a determinable reduction, amelioration, eliminationor prevention of hematophagous infestation in the mammal to which thevaccine of the present invention was administered. While the vaccines ofthe invention can be used with any mammal, of particular interest arelivestock, more particularly, horses, cattle, and the like. Thecompositions are useful for vaccination against the hematophagous fly ofthe suborder Cyclorrhapha, more particularly of the species Haematobiairritans, even more particularly of the subspecies irritans or exigua.However, the invention vaccination against any hematophagous organismwhere such vaccination using compositions and methods of the presentinvention is therapeutically effective.

For veterinary applications, the compositions of the invention can beformulated into any acceptable pharmaceutical preparation as describedbelow or any other acceptable preparation for veterinary use. In oneembodiment of the invention, the vaccines comprise therapeuticallyeffective amounts of the proteins of the invention, or any variant orfragment thereof as described herein.

In a preferred embodiment, the vaccines comprise the nucleotidecompositions of the invention as described herein. As described by Coxet al. (1993) J. Virol. 67:5664-5667; Fynan et al. (1993) Proc. Natl.Acad. Sci. USA 90:11478-11482; and Lewis et al. (1997) Vaccine15:861-864; and reviewed by Robinson (1997) Vaccine 15:785-787; andTighe et al. (1998) Immunol. Today 19:89-97, the contents of all ofwhich are herein incorporated by reference, nucleic acid vaccines can bereadily constructed and produced. In general, target DNA sequencesencoding the protein to be used as an immunogen are cloned intoeukaryotic expression vectors. The constructed plasmid is grown inbacteria and purified. The purified plasmid DNA is then directlyinjected into the animal generally by intramuscular injection, but alsoby intradermal injection; where its expression by cells in theinoculated host produces the target protein, thereby raising an immuneresponse. See, for example, Cox et al. (1993) J. Virol. 67:5664-5667,herein incorporated by reference. Nanogram levels of DNA-expressedprotein may be utilized to stimulate an immune response and protectagainst infectious agents achieved by skin, muscle and intravenousinoculations of DNA. See, for example, Fynan et al. (1993) Proc. Natl.Acad. Sci. USA 90:11478-11482; Cox et al. (1993) J. Virol. 67:5664-5667,herein incorporated by reference. Such plasmids introduced byintramuscular or intradermal injection stimulate a protective responsethat abrogates clinical disease following challenge.

The compositions of the present invention can be formulated intopharmaceutical preparations for therapeutic use as antithrombin agents.Such compositions find use in the treatment of venous thrombosis,vascular shunt occlusion and thrombin-included disseminatedintravascular coagulation.

The compositions of the invention can be used alone or in combinationwith other antithrombin and therapeutic agents including veterinaryagents such as vaccines. Other agents are known in the art.

The antithrombin compositions can be formulated according to knownmethods to prepare pharmaceutically useful compositions, such as byadmixture with a pharmaceutically acceptable carrier vehicle. Suitablevehicles and their formulation are described, for example, inRemington's Pharmaceutical Sciences 19th ed., Osol, A. (ed.), MackEaston Pa. (1980). In order to form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the antithrombin protein, eitheralone, or with a suitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achieved bythe use of polymers to complex or absorb the compositions. Thecontrolled delivery may be exercised by selecting appropriatemacromolecules (for example, polyesters, polyamino acids, polyvinylpyrrolidone, ethylene-vinylacetate, methylcellulose,carbosymethylcellulose, or protamine sulfate). The rate of drug releasemay also be controlled by altering the concentration of suchmacromolecules.

Another possible method for controlling the duration of action comprisesincorporating the therapeutic agents into particles of a polymericsubstance such as polyesters, polyamino acids, hydrogels, poly(lacticacid) or ethylene vinylacetate copolymers. Alternatively, it is possibleto entrap the therapeutic agents in microcapsules prepared, for example,by coacervation techniques or by interfacial polymerization, forexample, by the use of hydroxymethyl cellulose or gelatin-microcapsulesor poly(methylmethacrylate) microcapsules, respectively, or in a colloiddrug delivery system, for example, liposomes, albumin, microspheres,microemulsions, nanoparticles, nanocapsules, or in macroemulsions. Suchteachings are disclosed in Remington's Pharmaceutical Sciences (1980).

In more specific embodiments, a polypeptide of the invention may beconverted into a pharmaceutically acceptable salt. It may be convertedinto an acid additional salt with an organic or inorganic acid. Suitableacids include acetic, succinic and hydrochloric acid. Alternatively, thepeptide may be converted into a carboxylic acid salt such as theammonium salt or an alkali metal salt such as the sodium or potassiumsalt.

A polypeptide or pharmaceutically acceptable salt thereof may be used ina pharmaceutical composition, together with a pharmaceuticallyacceptable carrier or excipient therefore. Such a formulation istypically for intravenous administration (in which case the carrier isgenerally sterile saline or water of acceptable purity). A polypeptidecan therefore be used for the therapy and prophylaxis of thrombosis andthromboembolisms in a human or other mammal, including the prophylaxisof post-operative thrombosis, for acute shock therapy (for example forseptic or polytraumatic shock), for the therapy of consumptioncoagulopathics, in hemodialyses, haemoseparations and in extracorporealblood circulation. In one embodiment of the invention, the polypeptideor salt thereof can be coadministered with a plasminogen activator, suchas tissue plasminogen activator.

The dosage depends especially on the specific form of administration andon the purpose of the therapy or prophylaxis. The size of the individualdoses and the administration regime can best be determined by way of anindividual judgment of the particular case of illness; the methods ofdetermining relevant blood factors required for this purpose arefamiliar to the person skilled in the art. Normally, in the case of aninjection the therapeutically effective amount of the compoundsaccording to the invention is in a dosage range of from approximatelyfrom 0.005 or 0.01 to approximately 0.05 or 0.1 mg/kg body weight,preferably from approximately 0.01 to approximately 0.05 mg/kg bodyweight.

The administration is effected by intravenous, intramuscular orsubcutaneous injection. Accordingly, pharmaceutical compositions forparenteral administration in single dose form contain per dose,depending on the mode of administration, from approximately 0.4 toapproximately 7.5 mg of the compound according to the invention. Inaddition to the active ingredient these pharmaceutical compositionsusually also contain a buffer, for example a phosphate buffer, which isintended to keep the pH value between approximately 3.5 and 7, and alsosodium chloride, mannitol or sorbitol for adjusting the isotonicity. Thepreparations may be freeze-dried or dissolved. An antibacterially activepreservative may be included, for example from 0.2 to 0.3%4-hydroxybenzoic acid methyl ester or ethyl ester.

A composition for topical application can be in the form of an aqueoussolution, lotion or gel, an oily solution or suspension or afat-containing or, especially, emulsified ointment. A composition in theform of an aqueous solution is obtained, for example, by dissolving theactive ingredients according to the invention, or a therapeuticallyacceptable salt thereof, in an aqueous buffer solution of from e.g., pH4 to pH 6.5 and, if desired, adding a further active ingredient, forexample an anti-inflammatory agent, and/or a polymeric binder, forexample polyvinylpyrrolidone, and/or a preservative. The concentrationof active ingredients is from approximately 0.1 to approximately 1.5 mg,preferably from 0.25 to 1.0 mg, in 10 ml of a solution or 10 g of a gel.

An oily form of administration for topical application is obtained, forexample, by suspending the active ingredient according to the invention,or a therapeutically acceptable salt thereof, in an oil, optionally withthe addition of swelling agents, such as aluminum stearate, and/orsurfactants (tensides) having an HLB value (“hydrophilic-lipophilicbalance”) of below 10, such as fatty acid monomers of polyhydricalcohols, for example glycerin monostearate, sorbitan monolaurate,sorbitan monostearate or sorbitan monooleate. A fat-containing ointmentis obtained, for example, by suspending the active ingredient accordingto the invention, or a salt thereof, in a spreadable fatty base,optionally with the addition of a tenside having an HLB value of below10. An emulsified ointment is obtained by triturating an aqueoussolution of the active ingredient according to the invention, or a saltthereof, in a soft, spreadable fatty base with the addition of a tensidehaving an HLB value of below 10. All these forms for topical applicationcan also contain preservatives. The concentration of active ingredientis from approximately 0.1 to approximately 1.5 mg, preferably from 0.25to 1.0 mg, in approximately 10 g of base.

In addition to the compositions described above and pharmaceuticalcompositions analogous thereto that are intended for direct medicinaluse in the body of a human or a mammal, the present invention relatesalso to pharmaceutical compositions and preparations for medicinal useoutside the living body of humans or mammals. Such compositions andpreparations are used especially as anticoagulant additives to bloodthat is being subjected to circulation or treatment outside the body(for example haemoseparation). Such preparations, such as stocksolutions or alternatively preparations in single dose form, are similarin composition to the injection preparations described above; however,the amount of concentration of active ingredient is advantageously basedon the volume of blood to be treated or, more precisely, on its thrombincontent. Depending on the specific purpose, the suitable dose is fromapproximately 0.01 to approximately 1.0 mg of the activeingredient/liter of blood, although the upper limit may still beexceeded without risk as the agent is harmless even in relatively highamounts.

Experimental

Collection and Rearing of H. irritans

Pupae were shipped from the U.S.D.A. Livestock Insects ResearchLaboratory in Kerrville, Tex., on a biweekly basis and stored at 4° C.until needed. They were removed and placed in stainless steel cages(18″×18″×18″) at room temperature (21-22° C.) with 16:8 hours (L:D) topromote emergence of adults. An absorbent cotton pad was placed on topof each cage and used as a wick to supply fresh blood to adults on adaily basis.

Wild-caught adults collected from the University of Arizona dairy herdand from the Auburn University beef and dairy herds were used for someassays. They were transported to the laboratory within an hour ofcollection and maintained as above prior to experimentation.

Recovery of Salivary Glands

Both sexes of H. irritans are obligate blood feeders and their salivaryglands are similar in morphology and location in the body to stableflies (Stomoxys calcitrans) and tsetse flies (Glossina spp.) Thefollowing protocol was used for dissection of glands: (a) the fly was“knocked down” with humidified CO₂, passed briefly through a 70% ethanol(ETOH) bath, and then rinsed in deionized water; (b) it was placed on aclean glass slide in a drop of chilled 0.15M saline and the legs, wingsand head were removed. The thorax was split sagittally using a razorblade or scalpel; (c) the fly was then transferred to a fresh drop ofchilled saline in a watch glass or a small dish filled with paraffin.Using minute dissecting needles, the two halves of the thorax were thenpeeled back; (d) using forceps, the abdominal cuticle was pulled away,exposing the internal organs. The salivary glands were then teased awayfrom the gut tissue. The anterior end of the gut (the cardia) wasclipped and then gut-salivary gland assembly withdrawn by pulling itthrough the abdomen-thorax constriction; (e) the glands were then teasedaway from the gut, rinsed once in cold saline and transferred to anEppendorf® tube to be kept in ice for collection, and then frozen at−70° C.

Preparation of Salivary Gland Extracts

Salivary gland extracts (SGEs) were prepared as described by Cupp et al.(1993) J. Insect Physiol. 39:817-821, or by sonication. For the formermethod, glands were homogenized in a 1:1 mixture of 0.15 M NaCl solutionand 0.1% Triton X-100 was added to the thawed sample, which was thenrefrozen. Extracts were prepared by thawing the solubilized sample,vortexing it for 30 seconds and then centrifuging it at 14,000×g for 30seconds at 4° C. For the latter method, sonic disruption of glands wasobtained using 70% cycle and 70% power output of a Sonifier® 450sonication instrument (Branson Ultrasonics, Danbury, Conn.) for 2minutes. Eppendorf® tubes with glands were thawed and the contentsdisrupted by holding the tip of each tube to the base of the sonic probeimmersed in an ice bath to disperse heat. Salivary gland extracts weretransferred to a new tube following removal of cell fragments bycentrifugation at ≈12,000×g for 5 minutes at 4° C. The amount of proteinper individual gland was determined using a BCA protein assay kit(Pierce® Biotechnology. Inc., Rockford, Ill.). Initial measurement ofsoluble protein obtained from sonicated H. irritans salivary glands was0.54±0.09 μg/pair of glands for females and 0.63±0.02 μg/pair of glandsfor males.

Collection of Saliva

To determine antihemostatic activity attributable specifically tosalivary secretion, two methods were joined which have been usedpreviously for the buffalo fly (Kerlin and Hughes (1992) Med. Vet.Entomol. 6:121-126) and mosquitoes (Hurlbut (1966) Am. J. Trop. Med.Hyg. 15:989-993) to collect saliva from these insects. Adult flies, heldat room temperature, were starved for 24 hours to insure that secretionswere retained in the salivary glands and that all gut contents weredigested. The latter precaution is necessary since muscoid flies oftenregurgitate during feeding. The flies were then anesthetized withhumidified CO2 and their wings removed with microdissecting scissors.The dealated flies were then glued to applicator sticks so that theirmouth parts could be positioned into a capillary tube containing mineraloil. Just prior to this step, each fly was injected with 1 μl of 80 mMserotonin. The fly's proboscis was then inserted into the oil which,because of its difference in viscosity with saliva, served as acollecting medium for the serotonin-induced secretions. Salivationusually began within 30-60 seconds and the saliva could be easily seenas a clear aqueous droplet when it was expelled into the oil.

Gel Electrophoresis

Unless otherwise indicated, proteins were resolved on 15%polyacrylamide/SDS gels (SDS PAGE) by the method of Laemmli (1970)Nature 227:680-685, and visualized by silver staining (Bassam et al.(1991) Annal. Biochem. 196:80-83). Stained gels are scanned fordensitometry analysis of band migration and staining intensity (PersonalDensitometer® S.I. device, ImageQuaNT® computer software for Windows NT,Molecular Dynamics®, Inc., Sunnyvale, Calif.).

Proteins in Saliva

FIG. 1 depicts molecular weight comparison of proteins in saliva ofcolony (lane C) versus field-collected (Lane B) flies by relativemobility on SDS PAGE. Molecular weight standards in the 10-220 kDa rangeare shown in lanes A and D. A very similar profile is observed exceptfor the presence of a light band at ≈36 KDa in field-collected flies.However, the concentration of proteins in the saliva of the 30field-collected flies (B), as determined by relative intensity ofstaining of bands, exceeds that of corresponding bands in the saliva of84 colony flies (C). This difference was observed routinely onsilver-strained gels and indicates that field populations of H. irritansproduce greater concentrations of salivary proteins than do flies fromthis colonized strain.

Apyrase Activity

Apyrase activity in SGEs was tested using a standard assay (see Cupp etal. (1993) J. Insect Physiol. 39:817-821). This enzyme rapidly degradesadenosine triphosphate (ATP) and adenosine diphosphate (ADP) to themonophosphate, thereby eliminating a crucial chemical signal thatordinarily promotes platelet aggregation. Extracts were prepared fromwild caught male and female flies which were maintained on water for 48hrs prior to dissection. Activity in this enzyme in SGEs was marginallydetectable in H. irritans (2.59±0.21 milliUnits/pair of salivary glandequivalents). This lack of apyrase activity was also confirmed by theinability of H. irritans saliva to affect ADP-induced aggregation ofplatelets in bovine platelet-rich plasma (unpublished observations).Thus, apyrase activity was eliminated as a mechanism of hematophagy byH. irritans.

Erythema Activity

We evaluated the potential of H. irritans saliva to induce erythema,using intradermal injections of SGEs or by direct feeding of male andfemale flies on the shaved back of a New Zealand White rabbit. As acontrol, we also injected Simulium vittatum SGEs which produce apersistent erythema within 15 min of intradermal delivery (Cupp et al.(1994) Am. J. Trop. Med. Hyg. 50:235-240). A colonized strain of S.vittatum served as a source of salivary gland material (Bernardo et al.(1986) Ann. Entomol. Soc. Am. 79:610-621). No erythema was produced byeither male or female H. irritans saliva, whether injected as an SGE ordelivered by bite. Simulium vittatum SGE produced a visible erythemawithin 15 minutes. Thus, erythma activity was eliminated as a mechanismof hematophagy by H. irritans.

Other Vasodilative Activity

Studies were conducted to detect the presence of vasodilative activityin H. irritans SGEs or saliva using tension measurements of rat stomach(assay for prostaglandin) and rabbit aortic strips, with and withoutintact endothelium (see Ribeiro et al. (1992) Exp. Parasitol.74:112-116; Ribeiro et al. (1994) J. Med. Entomol. 31:747-753). Todetect bradykinin or histamine activity in H. irritans SGEs, the assayfollowed the procedure of Webster and Prado (1970) which uses thecontraction in vitro of guinea pig ileum as a direct bioassay of kininactivity. Normal responses to test substances (prostaglandin E2 for ratstomach strips and norepinephrine or acetyl choline for rabbit aorticstrips) were obtained, while H. irritans SGE showed no vaso-activity.Initially, collections of induced saliva did show activity in the ratstomach strip assay but this was lost when methysergide maleate wasincluded (Pertz and Eich (1992) Navnyn Schmiedebergs Arch. Pharmacol.345:394-401. This substance is a known inhibitor of serotonin, thecompound used to elicit salivation by the fly. The presence of activityin serotonin-induced saliva, but not in SGE, indicated that theserotonin activity in those samples was derived from the injectedcompound used to elicit salivation. Extraction of H. irritans SGE toenhance detection of prostaglandin activity confirmed the negativeresults of the earlier vasodilatory study. No salivary activity wasdetected in the guinea pig ileum assay for bradykinin or histamine.Thus, the tested vasodilative activities were eliminated as mechanismsof hematophagy by H. irritans. The inability of hornfly SGE to elicitvasodilation when injected intradermally into the shaved skin of NZWrabbits, in vivo, was confirmed using laser doppler perfusion imaging.

Anti-coagulant Activity

The re-calcification time assay was chosen to screen for anticoagulantactivity, as this general assay can detect inhibitors that attack at anyof the three major arms of the coagulation cascade, the extrinsicpathway, the intrinsic pathway and the final common pathway. Salivarygland extracts were prepared from both male and female H. irritans andfrom female S. vittatum. SGEs from the latter species were used as apositive control because the same re-calcification time assay had beenused previously to detect anticoagulant activity in that species (Abebeet al. (1994) J. Med. Entomol. 31:908-911). Salivary gland extracts offemale H. irritans were as potent as those of S. vittatum in delayingthe re-calcification time of standard plasma as shown in FIG. 2. MaleSGEs also delayed re-calcification time (data not shown). Comparableinhibition occurred in spite of the fact that measured protein contentswere 50% lower in extracts of H. irritans. Thus, this anti-coagulantactivity was the only anti-hemostatic activity detected for the hornfly, H. irritans.

Anti-hemostatic Specificity

The recalcification time assay can detect inhibition of any step in thecascade of reactions that ultimately lead to blood-clotting(coagulation), and thus it is a useful general test to screen for thepresence of an unknown inhibitor. Because blood-clotting is the resultof a series of reactions, horn fly saliva could delay clotting byinhibiting a specific step in the blood-clotting cascade or,alternatively, delay the normal rate of hemostasis by dissolving a clotafter it was formed (fibrinolytic activity).

For analytical purposes the clotting reactions are typically groupedinto three sub-pathways which are monitored by different clottingassays; i.e., the intrinsic (activated partial thromboplastin timetest=APTT), the extrinsic (prothrombin time test=PTT) and the finalcommon pathway (thrombin time=TT). Recalcification time, PTT, TT andAPTT assays are well known by those ordinarily skilled in the art. Forexample, see Biggs et al. ((1962) Human Blood Coagulation And ItsDisorders, 3rd ed., Blackwell Scientific Publications, Oxford) forrecalcification time assays, and Turgeon M. L. ((1993) ClinicalHematology. Theory and Procedures, 2nd ed., Little, Brown and Company,Boston) for APTT, PTT and TT assays. APTT II is a modification of theAPTT I test and is more sensitive.

Using these tests, several properties of horn fly anticlotting activitywere determined as shown in Table 1:1) Horn fly salivary gland extractsor saliva caused delay in clotting of all the tests, indicating that atleast one inhibitor is present that works in the final common pathway,i.e. after the formation of thrombin from prothrombin. 2) Saliva fromwild-type flies contains more inhibitor activity than saliva collectedfrom the same number of colony flies. 3) Inhibitor activity in colonyflies held for 48 hours after emergence is greater than at 24 hourspost-emergence.

TABLE 1 Delay in blood clotting by Haematobia irritans salivary glandextracts (SGE) or serotonin-induced saliva. Source # flies Type of Assay% of Control* SGE-colony 1 Recalcification 106 SGE-colony 2Recalcification 128 Saliva-colony (24 h) 4 Recalcification 143Saliva-colony (48 h) 4 Recalcification 175 Saliva-wild type 1Recalcification 127 Saliva-wild type 2 Recalcification 161 SGE-colony 1APTT-I 113 SGE-colony 2 APTT-I 149 Saliva-colony 1 APTT-I 112 SGE-colony1 APTT-II 144 Saliva-wild type 1 APTT-II 210 SGE-colony 1 PTT 120SGE-colony 2 PTT 140 Saliva-wild type 1 PTT 156 SGE-colony 1 TT NDSGE-colony 2 TT 109 Saliva-wild type 1 TT 158 ND not determined *Eachvalue is the mean of 4 assays

Inhibition of clotting in the TT assay by horn fly saliva indicates thata reaction occurring after the formation of thrombin is targeted. Tworeactions occur after that point —1) the formation of fibrin monomers bythe action of thrombin (factor II) on fibrinogen and 2) thecross-linking of fibrin monomers by the action of factor XIII. Thrombinis also involved in the activation of factor XIII. Thus, thrombin(factor II) was a probable target of horn fly saliva. To test thispossibility, clotting times of plasma that had been depleted of factorII by using specific antibodies (Sigma-Aldrich® Co., St. Louis, Mo.)were determined. Addition of increasing amounts of normal plasma,(containing factor II), decreased the time for clotting as measured bythe PTT assay (FIG. 3, −saliva). When horn fly saliva (equivalent to 2flies) was added with the increasing amounts of normal plasma (FIG. 3,+saliva), the percentage delay in clotting time increased withincreasing amounts of factor II (present in normal plasma). This patternindicated that saliva contained a specific inhibitor of factor II.

Thrombin clotting action can be measured using a synthetic substrate(S238, American Diagnostica® Inc., Greenwich, Conn.) that produces achromophore following hydrolysis by thrombin. The rates of hydrolysis ofS238 by bovine thrombin alone (250 pM; FIG. 4, −saliva) and in thepresence of horn fly saliva (equivalent to 2 flies) were measured atincreasing concentrations of substrate over the range of 2.5-100 μM.These data confirmed the observation that horn fly saliva contains aninhibitor of thrombin and indicate that it may be a competitiveinhibitor, as its effect is diminished when substrate is unlimited (100μM). Several models can account for such biochemical behavior (Segel(1976) Biochemical Calculations, John Wiley & Sons, New York). Foranalysis, a Dixon plot is generated by determining the velocity (v) ofsubstrate hydrolysis by thrombin in the presence of different fixedconcentrations of substrate, and plotting 1/v versus inhibitorconcentration. This provides the means to identify the type ofinhibition and to determine the inhibition constant, Ki.

Characterization of the Physical Properties of the Anti-ClottingComponent(s) in Horn Fly Salivary Glands to Devise a Purification Plan

APTT clotting times in Table 2 indicate that activity in SGE isdiminished after sitting at room temperature for 60 minutes or whensubjected to 100° C. for 5 minutes. The activity precipitates withethanol, and is reasonably stable to treatment with acetonitrile/TFA andlyophilization. These physical attributes are consistent with aproteinaceous inhibitor that can be purified under standard HPLCprocedures using acetronitrile/TFA gradient elution.

TABLE 2 Characterization of the physical properties of anti-clottingactivity in Haematobia irritans salivary gland extracts. Treatment APTTClotting Time (Seconds) Control 52.3 SGE-Time O 62.6 SGE-roomtemperature × 60 min 56.8 SGE-100° C. × 5 min 55.2 SGE-ethanolprecipitate 59.8 SGE-ethanol supernatant 50.1 SGE-lyopholized 57.0SGE-50% acetonitrile/0.1% TFA 57.8HPLC Purification and Recalcification Assay of HPLC Saliva Fractions

For analytical method development, saliva from 100 to 150 flies waspooled for each HPLC run. For preparative separation, saliva from morethan 500 flies was used for each run. Before injection onto the column,pooled saliva was always diluted with the initial solvent of the pairedgradient Åsolvents. A macrosphere, C18, 4.6×250 mm, 300 Å column(AllTech) was used for all H PLC preparations. Protein elution wasmonitored by UV absorption at 220 nM, which detects peptide bonds.Components eluted from the column were collected at 0.5 or 1 minuteintervals. An aliquot for activity assays was transferred from eachfraction to a second tube containing bovine serum albumin (BSA) beforelyophilization of all samples to remove organic solvents. Fractionsdried with BSA (used to increase solubilization of purified protein)were reconstituted with Tris buffer (5 mM tris, 150 mM NaCl, pH 7.4 at37° C.). Inhibitory activity in fractions was defined by the delay inclot formation using the above-described recalcification assay.

FIGS. 5A, 5B, and 5C depict the three reversed phase HPLC columnprocedures used to obtain a highly pure preparation of anti-clottingactivity. Black lines are HPLC chromatograms, while the gray barsindicate clotting times of recalcification assay. FIG. 5A shows HPLCseparation of H. irritans saliva using gradient elution (acetonitrile,2-propanol, and TFA). FIG. 5B shows HPLC separation of fraction withmaximum anticlotting activity in “A” using gradient elution(acetonitrile and TFA). FIG. 5C shows HPLC separation of fraction withmaximum anticlotting activity in “B” using gradient elution(acetonitrile and HCl). Clotting data from the first fractionation run(A) indicated that horn fly saliva contains only one clotting inhibitorthat elutes at approximately 45 minutes under the conditions used. Forsecondary HPLC separation, fractions from the target peak were combinedand injected directly onto the column after the column had beenequilibrated with the initial solvent. Anti-coagulant activity wasretained after 3 consecutive HPLC runs, vacuum drying, and storage for 4days at 4° C.

FIG. 6 shows SDS-PAGE of horn fly salivary anticlotting protein afterthe 3-step HPLC separation. Lane 1 contained protein concentrationmarker; lane 2, protein molecular weight standard marker; lane 3, theHPLC fraction with higher anticlotting activity (FIG. 5-C) and lane 4,the HPLC fraction with lower anticlotting activity (FIG. 5-C). Thisprofile indicated a single protein of high purity with a relativemobility of ≈16.5 KDa.

Construction of a Horn Fly Salivary Gland cDNA Library

Total salivary gland RNA (stored in several aliquots at −70° C. for aperiod of ≈3 years) was thawed, pooled and mRNA isolated using poly(A)Quick® reagents (Stratagene® Corporation, LaJolla, Calif.). A cDNAlibrary was constructed using a ZAP express™ vector and kit fromStratagene® Corporation. Preliminary analysis of numbers of insertsindicated that a relatively small number of primary inserts was obtained(≈3×10⁴). Approximately ⅕ of the primary library was reserved and theremainder used for one round of amplification to yield a titer of5.1×10⁶ plaque forming units (PFU) per ml.

Cloning the cDNA Coding for Thrombostasin

An estimated 110 pmoles of HPLC-pure thrombostasin were sent to HarvardMicrochemistry Lab to obtain a precise molecular mass by massspectroscopy and identification of 30 residues of the amino-terminal(N-) sequence. Although our analysis by SDS/PAGE consistently indicateda mass of ≈16.5 KDa (see, for example, FIG. 6), mass spectroscopy of theHPLC-pure sample detected an apparent “family” of 4 proteins with anaverage mass of 9.3±0.06 KDa. One N-terminal sequence was obtained fromthe ˜9 KDa protein (SEQ ID NO: 3), indicating that the variable masseswere obtained from largely identical proteins that may have variable ionpairs or that differ by as few as 1-2 amino acids. The sequence from theN-terminus also suggested that the protein is highly acidic. A secondsample of thrombostasin, which was purified by HPLC and sent foranalysis, yielded a similar mass. The unused remainder of this secondsample was re-analyzed by SDS/PAGE. Again, the protein ran as a ˜16.5mass. Search of the scientific literature revealed another report ofhighly acidic protein that produced an anomalously high molecular masswhen analyzed by PAGE (Takano et al. (1988) Biochemistry 27:1964-1972).In order to confirm the molecular mass, a third batch of thrombostasinwith confirmed activity in a re-calcification assay, was subjected toSDS/PAGE. The single band of ˜16.5 KDa protein was transferred to a PVDFmembrane. The blot was stained with ponceau S to reveal the transferredthrombostasin band. This band and a control region of similar area wasexcised and sent to the Harvard Lab for sequence analysis. TheN-terminal sequence from this analysis (SAGPI) confirmed the identity ofthe first 5 amino acids of the N-terminus.

The N-terminal sequence obtained from the first 30 residues ofthrombostasin as set forth in SEQ ID NO: 3 was used to constructdegenerate nucleotide primers by the Scott-Ritchey Research Center(SRRC) DNA lab at Auburn University. For template DNA, an aliquot of theHaematobia irritans salivary gland cDNA was used that had been removedand frozen at −20° C. following first strand cDNA synthesis for theabove-described library construction. A PCR reaction using thistemplate, the degenerate forward primer designed from thrombostasinN-terminal sequence and a reverse primer of oligo dT, yielded a productof approximately 350 base pairs. A 1 μl aliquot of the PCR product wasused in a ligation reaction with the PCR 2.1 vector (Invitrogen®Corporation, San Diego, Calif.) at 14° C. overnight. OneShot™ competentcells (Invitrogen® Corporation) were then transformed with the ligationproduct and transferred onto LB agar plates containing ampicillin.Following overnight growth, blue and white colonies were visiblerepresenting cells containing plasmid without an insert (blue) andplasmids with an insert that disrupted the beta-galactosidase gene(white colonies). Ten white and 2 blue colonies were picked foramplification in liquid culture by overnight growth at ˜30° C. Aliquotsof each culture were preserved by storage in glycerol at −70° C. Plasmidsize was estimated visually by ethidium bromide staining and comparisonto molecular weight markers. DNA minipreps were prepared and sequencedby the SRRC DNA lab using primers based on sequences in the plasmidvector flanking the multiple cloning insertion site.

Analysis of the deduced amino acid sequence of the protein, set forth inSEQ ID NO: 2, coded for by the PCR-cloned cDNA set forth in SEQ ID NO:1, confirmed identity to thrombostasin; i.e. the cDNA codes for a ˜9 KDaprotein and includes all the amino acids revealed by N-terminalsequencing, even though only a portion of that information was used inthe synthesis of degenerate primers that permitted amplification by PCR.Twenty-one percent of the putative protein is comprised of aspartic andglutamic acid residues. This information also confirmed that the cDNAencoding active thrombostasin is contained in the H. irritans cDNAlibrary. A search of protein databases in GenBank revealed no similarsequences.

Preparation of a Digoxigenin-Labeled Thrombostasin Probe

The above-described PCR-cloned thrombostasin cDNA fragment was used toproduce a digoxigenin-labeled probe for screening the H. irritans cDNAlibrary under very stringent conditions. A digoxigenin-labeled primerwas synthesized by PCR using the cloned thrombostasin fragment astemplate and the Genius™ system (Boehringer Mannheim® Corporation,Indianapolis, Ind.) in a 1:5 digoxigenin-11-dUTP to dTTP ratio. Thedigoxigenin-labeled DNA was purified by agarose gel electrophoresis.Yield of labeled probe was estimated by titration and visual comparisonto a DIG-labeled control DNA provided in the Genius™ Kit.

Cloning and Sequencing of a Full-Length cDNA

XL1 blue cells were transfected with 50,000 plaque forming units (pfu)from the amplified library and plated on a 150-mm NZY plate. Followingovernight incubation, the plate was chilled for 2 hr at +4° C. beforeplaque lifts made in duplicate with nylon membranes and probed with thedigoxigenin-labeled DNA fragment. In brief, “lifted” DNA was denaturedfor 5 min at RT, dried for 5 min, neutralized 5 min and cross linked ina Stratalinker® 1800 (Stratagene® Corp., La Jolla, Calif.) on autolinkcycle; pre-hybridization and hybridization was in 5×SSC, 0.1%N-lauroylsarcosine, 0.02% SDS, 2% blocking reagent and 50% formamide at65 EC; membranes were washed 4 times before visualization of thehybridized DIG-thrombostasin by incubation with anti-digoxigeninconjugated to alkaline phosphatase followed by substrate which producesa blue colored product. Several plaque picks from the first screeningwere subcloned to confirm positive clones in a secondary screen. Phagewas extracted from the plaque picks in SM buffer and amplified by growthin XL1-Blue MRF cells on NZY plates as described above. DNA was isolatedin minipreps of bacterial colonies grown overnight. Positive clones weretested by PCR amplification with thrombostasin-specific forward andreverse internal primers which were synthesized based on sequence in thecloned PCR fragment. Positive clones were further tested by anadditional plaque assay and shown to be pure by hybridization of allcolonies with the DIG/labeled probe.

Phagemids containing cloned inserts were obtained by automatic excisionusing the ExAssist®/XLOLR system and protocol of Stratagene® Corp.Colonies were grown on LB-kanamycin plates and glycerol stocks preparedfor storage at −70° C. Similar colonies were picked for amplification byovernight growth. DNA was extracted in minipreps and analyzed byautomated cycle sequencing (SRRC) in the forward direction using primersT3 and thrombostasin-F1 and in the reverse directions using primers T7and thrombostasin-R1, and with forward and reverse primers to sequencesinternal to the termini. Several cDNA clones were obtained andsequenced. The nucleotide sequences for a partial cDNA designated TB8are set forth in SEQ ID NO: 4, and the amino acid sequence encodedtherein are set forth in SEQ ID NO:5. It is noted that amino acidresidues 88-168 set forth in SEQ ID NO:5 encoded by nucleotides 263-505set forth in SEQ ID NO:4 correspond to active thrombostasin.

The Wisconsin Package™ of the Genetics Computer Group (GCG, MadisonWis.) was used to analyze nucleic acid and putative protein sequences ofthrombostasin cDNA.

To obtain the full length cDNA sequence, a 5′ RACE (Rapid Amplificationof cDNA Ends) procedure was employed, utilizing salivary gland mRNA andinternal primers having 3′ consensus sequence corresponding to the cDNAclones described above. Overlapping sequences were compiled to determinea composite full length cDNA sequence. The cDNA clone TB8 describedabove was used to construct a full length cDNA encoding thrombostasin,by adapting the clone to contain the 5′ end nucleotides determined fromthe 5′ RACE procedure. The nucleotide sequences for the full length cDNAare set forth in SEQ ID NO:6 and the amino acid sequences encodedtherein is set forth are SEQ ID NO:7. It is noted that amino acidresidues 95-175 set forth in SEQ ID NO: 7 encoded by nucleotides 283-525set forth in SEQ ID NO:6 correspond to active thrombostasin.

Production of a Recombinant Thrombostasin (r-thrombostasin) Protein

Thrombostasin plasmid DNA and the transfer vector pBacPAK8 (CLONTECH®,Palo Alto, Calif.) were digested with 2 restriction enzymes that cut inthe plasmid's multi-cloning sites but not internal sequences ofthrombostasin. Excised thrombostasin and linearlized pBacPAK8 werepurified by TAE gel electrophoresis. Digested bands were excised and DNAextracted with Sephaglas® Band Prep Kit (Pharmacia Biotech™, Uppsala,Sweden). A 1:2 (vector:insert) ligation reaction was setup to runovernight at 15° C. OneShot™ cells were transformed with the BacPAK8plasmid containing the thrombostasin insert as described for the PCRfragment. Transformed cells were grown overnight on LB ampicillinplates. Several colonies were selected for liquid, overnight growth at37° C. Glycerol stocks were prepared and frozen at −70° C. and plasmidquick preps made for size evaluation by agarose gel visualization.Miniprep DNA was prepared by column purification (Qiagen® Corp., SantaClarita, Calif.) for DNA sequencing using the Bac 2 primer (CLONTECH®Laboratories. Inc.).

A recombinant baculovirus containing the thrombostasin insert wasgenerated by co-transfection of Sf9 cells with BacPAK8/thrombostasinplasmid TB8/3 and Bsu36I digested BacPAK6 viral DNA using Lipofectin™transfection reagent (Life Technologies™, Grand Island, N.Y.) and HighFive™ Serum-Free Medium (Invitrogen® Corp., Carlsbad, Calif.). Controlsincluded wild type virus (positive control) and plasmid DNA only(negative control). Cells were incubated with transfection medium for 5hr at room temperature before adding TNM-FH medium containing 10% fetalbovine serum (TNM-FH/FBS), and further incubated at 27° C. for 72 hrs.Cell culture supernatant containing virus was collected and stored at 4°C. A plaque assay was performed to isolate pure thrombostasin-virusclones from the cell supernatant. Thirty-five nun plates containing1.5×10⁶ Sf9 cells each were infected in duplicate with 100 μl ofsupematant or a dilution up to 10 ⁻³ in a 100 μl volume of TNM-FH/FBSmedium. After sitting for 1 hr at room temperature, infection medium wasremoved and the cells overlaid with 3.5 ml each of Grace's medium (LifeTechnologies™, Grand Island, N.Y.), containing 10% FBS, 50 μg/mlGentamicin and 1% agarose. Cells were incubated in a plastic storage boxwith moist paper towels at 27° C. After 5 days, a second overlay wasadded that also included 0.16 mg/ml neutral red dye and 250 μg/ml Xgal.After the agarose overlay formed a gel, the dishes were inverted andincubated for 48 hr at room temperature. Clear, positive plaques werepicked and virus eluted by incubation overnight in TNM-FH medium. Sf9cells were infected with eluted virus and incubated for 4 days at 27° C.to generate passage 1 virus.

Cells were collected in phosphate buffered saline (10 mM, pH 7.4) andDNA extracted using the Stratagene® Corporation DNA micro extraction kitand protocol II in the instruction manual. Extracted DNA was used astemplate for PCR with a thrombostasin forward and reverse primer pairand the Bac1/Bac2 primer pair (CLONTECH® Laboratories, Inc.).Amplification with both primer pairs assured that the correcttransformation event occurred. A secondary plaque assay was conducted toassure clone purity, and to determine virus titer.

Characterization of r-thrombostasin Production

Sf9 cells were infected with virus (multiplicity of infection=2) andincubated at 27° C. until media are collected at 12, 24, 48, 72 and 96hr. Virus is concentrated and removed from media by centrifugation inCentriplus™ 100 concentrators (Amicon® Corporation, Beverly, Mass.) at3,000×g for 2 hr at room temperature. Total protein in the <100 KDafraction is estimated by the modified Lowry Assay (Sigma-Aldrich® Co.,St. Louis, Mo.).

Anti-clotting and/or antithrombin activity of r-thrombostasin is testedusing the chromogenic substrate S238 assay as described above.

Purification of r-thrombostasin by RP/HPLC

Large molecular weight components (≧10 KDa) in the virus-free cellculture supernatant are concentrated by centrifugation at 3,000×g for 4hr in Centriplus™ 10 microconcentrators. RP/HPLC using a C18 macrospherecolumn and elution with an acetonitrile gradient is used for isolationof r-thrombostasin from other medium components as described above.

Immunogenic properties of thrombostasin in a laboratory animal model(rabbits) as the first step toward production of a nucleic acid (DNA)vaccine against horn fly blood-feeding.

Anti-hemostatic proteins in the saliva of blood-feeding insects are nothighly immunogenic (see Cupp and Cupp (1997) J. Med. Entomol. 34:87-94).This experimental observation agrees with the intuitive concept thatgeneration of an immune response, especially production of neutralizingantibody, might prevent or decrease blood feeding and production ofprogeny and fitness. Thus, it is important to develop methods to elicita robust immune response to such molecules. Moreover, effectiveimmunization of cattle in the field also requires a practical vaccinethat needs a minimum of handling and storage. In the past few years,immunization with nucleic acids has been demonstrated to generate strongimmune responses to encoded proteins that can be directed to specificimmune compartments by the location and/or amount of nucleic acidadministered. Such vaccines, composed of plasmids with the DNA ofinterest inserted, can be produced at low cost and by relative simpletechniques of bacterial culture; they are stable to storage at roomtemperature and thus circumvent many of the problems of protein-basedvaccines. Thus, initially, immunization of rabbits is tested withthrombostasin nucleic acid.

A vaccine plasmid is constructed containing the CMV promoter andkanamycin resistance for selection. The procedures for restrictiondigestion and re-ligation of the baculovirus transfer vector asdescribed above is used to produce the thrombostasin containing vaccineplasmid (TS-Vac).

Serum, serving as pre-immunization control, is obtained from bloodsamples taken from each rabbit via the large ear vein. Nine rabbits areinjected intradermally with one of three TS-Vac plasmids in PBS (500 μgof plasmid with no insert=control; 200 μg, or 500 μg TS-Vac=testplasmids). Blood is sampled after approximately 4 weeks and tested forhumoral (the presence of specific antibody titer) and cellular response(blastogenic response) to thrombostasin. These parameters are monitoredon a weekly basis thereafter up to 20 weeks post-injection. Allimmunological assays used are standard and have been used in previouslypublished work on immune response to salivary factors of blood-feedinginsects (Cross et al. (1993) J. Med. Entomol. 30:725-734; Cross et al.(1993) J. Med. Entomol. 30:928-935).

Antibody titer is determined by a direct ELISA assay (Cross et al.(1993) J. Med. Entomol. 30:725-734; Cross et al. (1993) J. Med. Entomol.30:928-93 5). Briefly, 96 well flat-bottomed microtiter plates arecoated with r-thrombostasin, or non-specific protein, then blocked with1% bovine serum albumin (BSA) in PBS. Wells are incubated withpre-immune sera or test sera, then washed with PBS-tween before additionof alkaline phosphatase-conjugated anti-rabbit IgG (Sigma-Aldrich® Co.,St. Louis, Mo.) or anti-rabbit IgM (Southern Biotechnology Assoc. Inc.,Birmingham, Ala.) Following substrate reaction with p-nitrophenylphosphate (pNPP), color intensity is read at 405 nm using a Spectramax®microtiter plate reader (Molecular Devices® Corporation, Sunnyvale,Calif.). Antibody titer is calculated and compared among treatments todetermine the optimum amount of immunogen for antibody generation.

Antibody is evaluated for specificity to thrombostasin by dot blot(Cross et at. (1993) J. Med. Entomol. 30:725-734) using r-thrombostasin.R-thrombostasin, or bovine serum albumin (BSA) control is dotted ontowells of 96-well nitrocellulose-bottomed microtiter plates (Millipore®Corporation, Marlborough, Mass.). Non-fat milk (3%) in PBS is used as ablocking solution. Test sera are added and incubated for 1 hr beforewashing and substrate reaction. Specificity of reaction is determined byvisual inspection.

Cellular response to thrombostasin is tested using peripheral bloodmononuclear cells (PBM) isolated by Ficoll-Paque centrifugation of bloodcollected in EDTA as anticoagulant (Ramachandra & Wikel (1992) J. Med.Entomol. 29:818-826). Viability of isolated cells is determined on analiquot using fluorescein diacetate (FDA; Sigma-Aldrich® Co., St. Louis,Mo.) which brightly labels healthy cells. PBM are added at 5×10⁵cells/well of a microtiter plate before addition of r-thrombostasin,horn fly saliva or non-specific protein. The mitogen ConA is added andincubation continued for 72 hr. Cellular response to test protein isdetermined using the MTT colorimetric assay (Denizot and Land (1986) J.Immunol. Methods. 89(2):271-277) which is read in the Spectramax®microtiter plate reader (Molecular Devices® Corporation. Sunnyvale,Calif.) at 570 nm. Blastogenic response to thrombostasin is determinedby increase over control. Comparison of response in the presence ofwhole saliva can monitor for immunomodulating factors in saliva.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Other modifications and embodiments of the invention will come to mindin one skilled in the art to which this invention pertains having thebenefit of the teachings presented herein. Therefore, it is to beunderstood that the invention is not to be limited to the specificembodiments disclosed. Although specific terms are employed, they areused in generic and descriptive sense only and not for purposes oflimitation, and that modifications and embodiments are intended to beincluded within the scope of the appended claims.

1. A purified protein having antithrombin activity, wherein said proteincomprises an amino acid sequence that is at least 80% identical to theamino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:5, or SEQ IDNO:7.
 2. The protein of claim 1, wherein said protein comprises an aminoacid sequence that is at least 90% identical to the amino acid sequenceset forth in SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:7.
 3. The protein ofclaim 2, wherein said protein comprises the amino acid sequence setforth in SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:7.
 4. The protein ofclaim 1, wherein said protein is produced by recombinant methods.
 5. Theprotein of claim 1, wherein said protein modulates the immune response.6. A pharmacological composition comprising a therapeutically effectiveamount of the protein of claim
 1. 7. A purified protein havingantithrombin activity, wherein said protein comprises an amino acidsequence that is at least 95% identical to the amino acid sequence setforth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO:
 7. 8. The protein ofclaim 7, wherein said protein is produced by recombinant methods.
 9. Theprotein of claim 7, wherein said protein modulates the immune response.10. A pharmacological composition comprising a therapeutically effectiveamount of the protein of claim
 7. 11. A purified protein that hasantithrombin activity, wherein said protein comprises an amino acidsequence that comprises at least 15 contiguous amino acids of thesequence set forth in SEQ ID NO:2, SEQ ID NO: 5, or SEQ ID NO:
 7. 12.The protein of claim 11, wherein said protein comprises an amino acidsequence that comprises at least 15 contiguous amino acids of thesequence set forth in SEQ ID NO:7 between amino acid residue 95 andamino acid residue
 175. 13. The protein of claim 11, wherein saidprotein comprises an amino acid sequence that comprises at least 30contiguous amino acids of the sequence set forth in SEQ ID NO:2, SEQ IDNO: 5, or SEQ ID NO: 7.